Aggregation of alpha synuclein has strong implications in Parkinson’s disease. The heterogeneity of folding/aggregation landscape and transient nature of the early intermediates result in difficulty in developing a successful therapeutic intervention. Here we used fluorescence measurements at ensemble and single molecule resolution to study how the late and early events of alpha synuclein aggregation modulate each other. In-vitro aggregation data was complemented using measurements inside live neuroblastoma cells by employing a small molecule labeling technique. An inhibitor molecule (arginine), which delayed the late event of amyloidosis, was found to bind to the protein, shifting the early conformational fluctuations towards a compact state. In contrast, a facilitator of late aggregation (glutamate), was found to be excluded from the protein surface. The presence of glutamate was found to speed up the oligomer formation at the early stage. We found that the effects of the inhibitor and facilitator were additive and as a result they maintained a ratio at which they cancelled each other’s influence on different stages of alpha synuclein aggregation.

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a b-sandwich protein containing 11 b strands and a very short stretch of 3 10 helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete b-sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV-CD and FTIR spectra clearly suggest that the low-pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete b-sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non-native helical propensity of the protein structure.

Binding between AuNC and Mycobacterium tuberculosis derived protein.

Although significant efforts have been devoted to develop nanoparticle-based biopharmaceuticals, it is not understood how protein conformation and nanoparticle surface modulate each other in optimizing the activity and/or toxicity of the biological molecules. This is particularly important for a protein, which can adopt different conformational states separated by a relatively small energy barrier. In this paper, we have studied nanoparticle binding-induced conformational switch from β-sheet to α-helix of MPT63, a small major secreted protein from Mycobacterium tuberculosis and a drug target against Tuberculosis. The binding of magnetite nanoparticles to MPT63 results in a β-sheet to α-helix switch near the sequence stretch between the 19th and 30th amino acids. As a consequence, the immunogenic response of the protein becomes compromised, which could be restored by protein engineering. This study emphasizes that conformational stability toward NP surface binding may require optimization involving genetic engineering for development of a nanoparticle conjugated pharmaceutical.

Fluorescent metal nanoclusters have generated considerable excitement in nanobiotechnology, particularly in the applications of biolabeling, targeted delivery, and biological sensing. The present work is an experimental and computational study that aims to understand the effects of protein environment on the synthesis and electronic properties of gold nanoclusters. MPT63, a drug target of Mycobacterium tuberculosis, was used as the template protein to synthesize, for the first time, gold nanoclusters at a low micromolar concentration of the protein. Two single cysteine mutants of MPT63, namely, MPT63Gly20Cys (mutant I) and MPT63Gly40Cys (mutant II) were employed for this study. The experimental results show that cysteine residues positioned in two different regions of the protein induce varying electronic states of the nanoclusters depending on the surrounding amino acids. A mixture of five-atom and eight-atom clusters was generated for each mutant, and the former was found to be predominant in both cases. Computational studies, including density functional theory (DFT), frontier molecular orbital (FMO), and natural bond orbital (NBO) calculations, validated the experimental observations. The as-prepared protein-stabilized nanoclusters were found to have applications in the imaging of live cells.